With elderly population progressively increasing, a rapid growth of demential patients has become a serious social problem. Alzheimer's disease (AD) is a kind of dementia, and account for about 30% of senile dementia in Japan and more than half in Europe and U.S. AD is a kind of neurodegenerative disease, and its pathological characters include: (1) senile plaques in which A β is accumulated as a principle component are observed between neuronal cells; (2) abnormal phosphorylated tau protein aggregates in neuronal cells and fibrotic neurofibril are observed; (3) the cerebrum shrinks (deciduation of cerebral neocortex and neuronal cells of hippocampal). As the clinical characters, AD is a progressive dementia presenting hypergasia in general cognition, notably disorder of memory.
AD is classified into familial Alzheimer's disease (FAD), which is small in the number of cases and shows autosomal dominant inheritance, and sporadic Alzheimer's disease (SAD), which apparently lacks family medical history and accounts for 90% of the total Alzheimer's disease. As genes causative of FAD, are identified amyloid precursor protein (APP) gene located on chromosome 21, presenilin-1 (PS1) gene found on chromosome 14 and presenilin-2 (PS2) gene located on chromosome 1. The mechanism of development of FAD has been gradually becoming clear. On the other hand, SAD which makes up a majority of AD cases presents the same neuropathological observations as FAD, but its development mechanism is unknown in a considerable number of aspects.
For common clinical diagnosis of AD, cognition tests such as SM-IV, NINCDS-ADRDA and the like which are proposed in the United State are utilized. However, it is difficult to diagnose extremely slight cognition impediment in an early stage of AD as dementia using conventional cognition tests. The currently used definite diagnosis of AD consists of recognizing deposition of amyloid protein (senile plaques) and accumulation of tau protein (neurofibrillary tangles) in postmortem brain. Thus effective antemortem early diagnostic methods have not been established. In the present circumstances, it is too late when typical symptoms of AD (specific demential symptoms such as incapability of cognition and the like) are recognized.
At present, several kinds of anti-dementia medicines are distributed in a lot of countries, and donepezil has been clinically used in Japan since 1999. These medicines often have a beneficial effect on early-stage cases. Since the medicines are expected to exhibit beneficial effect if AD is diagnosed early, there is a demand for development of a diagnostic marker effective for early treatment of AD.
It has been reported that mRNA of a splicing variant (PS2V) of a PS2 gene with deletion of its fifth exon are observed manifesting frequently in about 70% of the encephala of SAD patients (J. Neurochem., Vol. 72, No. 6, 1999, 2498-2505). The mRNA of PS2V codes for a protein consisting of 124 amino acids having 5 amino acids (Ser-Ser-Met-Ala-Gly) (SEQ ID NO:7) added to 119 amino acid residues (Met1 to Leu119) at the N tenninal of PS2.
Immunohistologic detection of PS2V using samples of CA1 regions of the hippocampi of the encephala of SAD patients have confirmed 100% manifestation of PS2V (J. Biol Chem, 2001 Jan. 19; 276(3):2108-2114).
In vitro analysis has shown that {circle around (1)} in human neuroblastoma SK-N-SH cells in which PS2V is forced to expressed, susceptibility to endoplasmic reticulum (ER) stress increases since the induction of stress responsive protein GRP78 is suppressed; that {circle around (2)} PS2V inhibits autophosphorylation of Ire1 protein (ER stress sensor) and inactivates ER stress response, thereby causing the suppression on the expression of GRP78; and that {circle around (3)} in cells expressing PS2V, the production of both Aβ1-40 and Aβ1-42 is increased. From these three points, it is considered that in SAD, the expression of PS2V may possibly trigger neuronal death and increasing of Aβ production.
Accordingly, it is considered that highly sensitive detection of PS2V leads to early diagnosis of AD since the expression of PS2V plays an important role in AD development.
As the detection of PS2V, mention may be made of the detection of PS2V itself and the detection of mRNA of PS2V. For early diagnosis, it is impossible to collect a brain tissue of the encephalon of a patient. Therefore, it is necessary to carry out a test using a body fluid of a patient such as cerebrospinal fluid, blood, serum, urine or the like which can be collected relatively easily. However, mRNA is retained in cerebrospinal fluid or serum only for such an extremely short time and is decomposed so quickly that the diagnosis by detecting mRNA is impossible. On the other hand, PS2V is retained in cerebrospinal fluid or serum for a long time as compared with mRNA, and therefore, it is considered suitable for the diagnosis. However, since only a trace amount of PS2V is present in cerebrospinal fluid or serum, a method of detecting PS2V with high sensitivity is required.
Japanese Unexamined Patent Application Publication NO. 2000-37192 has already disclosed a method of producing PS2V by inducing the expression of an abnormal splicing varient of the PS2 gene using oxidative stress loading and β-amyloid stimulation in a culture system of neuronal cells. However, highly sensitive detection of PS2V has not been found.
Conventionally, alkaline phosphatase (referred to as ALP hereinafter) is often used as a labeling molecule in enzyme immunoassay and nucleic acid detection. Since ALP hydrolyzes a substrate which is a phosphate ester, the quantity of an object biomolecule conjugated with ALP can be determined by determining the quantity of a product obtained by hydrolysis with ALP.
Depending upon different determination techniques for the product, the detection of the product is classified into four types, i.e., absorptiometric detection, chemoluminescent detection, fluorescent detection and electrochemical detection.
Literature such as Analytica Chemica Acta 393 (1999) 95-102 and others have reported electrochemical detection using p-methoxyphenyl phosphoric acid as a substrate for ALP. However, methods of highly sensitive detection of PS2V using these substrates have not been found.
Accordingly, there are desired a method of highly sensitive detection of PS2V which is applicable to early diagnosis of AD and a substrate for a conjugating enzyme usable for the method.